rad51 mediates resistance of cancer stem cells to parp ......cancer therapy: preclinical rad51...

Cancer Therapy: Preclinical

RAD51 Mediates Resistance of Cancer Stem Cellsto PARP Inhibition in Triple-Negative BreastCancerYajing Liu1, Monika L. Burness1, Rachel Martin-Trevino1, Joey Guy1, Shoumin Bai2,Ramdane Harouaka1, Michael D. Brooks1, Li Shang1, Alex Fox1, Tahra K. Luther1,April Davis1, Trenton L. Baker1, Justin Colacino1, Shawn G. Clouthier1, Zhi-ming Shao3,4,Max S.Wicha1, and Suling Liu5

Abstract

Introduction: PARP inhibitors have shown promising resultsin early studies for treatment of breast cancer susceptibility gene(BRCA)–deficient breast cancers; however, resistance ultimatelydevelops. Furthermore, the benefit of PARP inhibitors (PARPi) intriple-negative breast cancers (TNBC) remains unknown. Recentevidence indicates that in TNBCs, cells that display "cancer stemcell" properties are resistant to conventional treatments, mediatetumor metastasis, and contribute to recurrence. The sensitivity ofbreast cancer stem cells (CSC) to PARPi is unknown.

Experimental Design: We determined the sensitivity of breastCSCs to PARP inhibition in BRCA1-mutant and -wild-type TNBCcell lines and tumor xenografts. We also investigated the role ofRAD51 in mediating CSC resistance to PARPi in these in vitro andin vivo models.

Results: We demonstrated that the CSCs in BRCA1-mutantTNBCs were resistant to PARP inhibition, and that these cells hadboth elevated RAD51 protein levels and activity. Downregulationof RAD51 by shRNA sensitized CSCs to PARP inhibition andreduced tumor growth. BRCA1–wild-type cells were relativelyresistant to PARP inhibition alone, but reduction of RAD51sensitized both CSC and bulk cells in these tumors to PARPitreatment.

Conclusions: Our data suggest that in both BRCA1-mutantand BRCA1–wild-type TNBCs, CSCs are relatively resistant toPARP inhibition. This resistance is mediated by RAD51, sug-gesting that strategies aimed at targeting RAD51 may increasethe therapeutic efficacy of PARPi. Clin Cancer Res; 23(2); 514–22.�2016 AACR.

IntroductionTriple-negative breast cancer (TNBC) is an aggressive subtype of

breast cancer that accounts for 10% to 17% of all breast cancers(1). A subset of TNBCs develops in BRCA1mutation carriers or inbreast cells that lose functional BRCA1 activity. Because BRCA1functions in DNA repair, synthetic lethality strategies involving

the use of DNA repair inhibitors have been developed. Theseinclude inhibitors of PARP, an enzyme that detects single-strandbreaks and recruits DNA repair molecules. Inhibition of PARPresults in accumulation of DNA breaks, which are recognized andrepaired by the DNA double-strand break pathway; thus inpatients with loss of BRCA activity, cells are subjected to celldeath due to excessive DNA damage. Successful implementationof a synthetic lethality therapeutic strategy was demonstrated inpreclinical models where PARP inhibition was shown to selec-tively target breast cancer cells lacking functional BRCA1 orBRCA2 (2–4).Olaparib is a potent inhibitor of PARP1 and PARP2and has been shown to potentiate the effects of DNA-damagingagents (5). Early clinical studies suggest that olaparib has con-siderable activity in patients with BRCA-deficient breast tumors(4, 6–9). However, the durability of this response and long-termclinical benefits of this approach have yet to be demonstrated.

There is increasing evidence that resistance to chemotherapeu-tic agents is mediated by a cellular subset that displays stem cellproperties. These cancer stem cells (CSC) are functionally definedas cells that have self-renewal capacity, as well as the capacity toregenerate a phenotypically similar bulk tumor. CSCs mediatetumor metastasis and contribute to chemotherapy and radiother-apy resistance. Furthermore, these therapies often increase levelsof CSCs in tumors. However, it is unknown whether the sameeffect occurs with targeted therapies, including PARP inhibitors(PARPi). In previous studies, we have reported that breast CSCsdisplay increased expression of someDNA repair genes compared

1University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan.2Department of Oncology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen Uni-versity, Guangzhou, China. 3Key Laboratory of Breast Cancer in Shanghai,Department of Breast Surgery, Fudan University Shanghai Cancer Center,Shanghai, China. 4Department of Oncology, Shanghai Medical College, FudanUniversity, Shanghai, China. 5Innovation Center for Cell Signaling Network andthe CAS Key Laboratory of Innate Immunity and Chronic Disease, School of LifeScience and Medical Center, University of Science and Technology of China,Hefei, China.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Y. Liu and M.L. Burness contributed equally to this article.

Corresponding Authors: Suling Liu, University of Science and Technology ofChina, 443 Huangshan Rd., Hefei, Anhui 230027, China. Phone/Fax: 086-551-63600773; E-mail: [email protected]; or Monika L. Burness, University ofMichigan Comprehensive Cancer Center, NCRC 26-319S, SPC 2800, 2800 Ply-mouth Rd., Ann Arbor, MI 48109-2800. Phone: 734-763-0807; Fax: 734-763-2202; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-1348

�2016 American Association for Cancer Research.

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with bulk tumor cells (10). We identified RAD51 as a potentialmediator of CSC resistance to PARPi. RAD51 forms a complexwith BRCA1, and this complex mediates homologous recombi-nation during DNA damage repair. This process is highly con-served throughout the phylogenetic tree. The association betweenelevated RAD51 levels and therapy resistance in cancer patientshas been shown in a wide range of cancer types including breastcancer (11). CSCs in several tumor types have been reported todisplay increased efficiency in DNA damage repair (12–15),suggesting that CSCs may be insensitive to PARP inhibition. Inthe present study, we utilized in vitro and in vivomodels of BRCA1-mutant and –wild-type TNBC to study the sensitivity of CSCs toPARPi and the role of RAD51 in mediating this response.

Materials and MethodsCell culture

TNBC lines SUM149 and SUM159 (a gift from Dr. StephenEthier, Karmanos Cancer Institute) were maintained in Ham's F-12 (Invitrogen) supplemented with 5% FBS (Hyclone), 1X anti-biotic-antimycotic, 10 mg/mL gentamicin (both from Life Tech-nologies), 5 mg/mL insulin, and 1 mg/mL hydrocortisone (bothfrom Sigma-Aldrich). MDA-MB-231 and HCC1937 were pur-chased from the ATCC and grown in DMEM and RPMI 1640medium (Invitrogen), respectively, with 10% FBS (Hyclone), 1%antibiotic-antimycotic, and 10 mg/mL gentamicin (both from LifeTechnologies). All cells were incubated at 37�C with 5% CO2.

Constructs and virus infectionThe inducible TRIPZ-shRNAswere purchased fromDharmacon

Open Biosystems (http://dharmacon.gelifesciences.com/open-biosystems/) to produce Tet-inducible RAD51 knockdown (KD)lentiviruses by transfecting 293T cells in the University of Michi-gan Vector Core Facility. The TNBC cells were infected with thepresence of polybrene (8 mg/mL; Millipore) overnight, and thenext day, medium containing viruses was washed and replacedwith fresh medium. Puromycin (Invitrogen) selection was per-formed for 7 days.

RNA extraction and real-time RT-PCRTotal RNA was extracted using an RNeasy Mini kit (Qiagen),

and 1 mg of RNA was used for making cDNA with the High-

Capacity cDNA Reverse Transcription Kit (Life Technologies).cDNA was analyzed using real-time quantitative RT-PCR assaysin an StepOne Real-Time PCR system (Applied Biosystems).RAD51 and GAPDH primers were obtained from Applied Biosys-tems. For the DNA repair PCR array, RNA was extracted using anRNeasy Mini kit (Qiagen), and total RNA was quantified byNanodrop (Thermo Fisher Scientific). RNA quality and integritywere assessed on the Agilent 2100 Bioanalyzer. cDNA was syn-thesized from 400 ng total RNA, loaded to the RT2 Profiler qPCRHuman DNA Repair Array (Qiagen), and amplified on a 7900HTReal-Time PCR system (Applied Biosystems) according to themanufacturer's instructions (Qiagen). Relative expression levelswere calculated based on the DDCt method as described in theRT2 Profiler PCR Array Handbook (06/2013; Qiagen).

Western blottingWestern blotting was performed to test the efficiency of RAD51

KD and overexpression. Infected cells with or without (control)doxycycline (Sigma-Aldrich) treatment were lysed in radioimmu-noprecipitation assay buffer (Sigma-Aldrich) containing proteaseand phosphatase inhibitors (Thermo Scientific). Note that 40 mgextracts from each sample was electrophoresed on a 4% to 12%Bis-Tris gel and transferred to a polyvinylidene difluoride mem-brane (Life Technologies). RAD51, BRCA1 (Cell Signaling Tech-nology), ALDH1A1 (LSBio), and horseradish peroxidase (HRP)–conjugated b-actin (Sigma-Aldrich) were applied overnight at 4�Cor 2 hours at room temperature in 5% non-fat milk (Bio-Rad)followed by secondary antibody anti-mouse-HRP or anti–rabbit-HRP (Cell Signaling). The membrane was stripped betweenantibodies using Restore Western Blot Stripping Buffer, and thestaining was detected by Super Signal West Pico Chemilumines-cent Substrate (Pierce).

Cell treatmentTo study RAD51 activity, cells were irradiatedwith a single dose

of 4Gywith an IC-320 orthovoltage irradiator (KimtronMedical)and then fixed at different time points after radiotherapy treat-ment. MTT assay was conducted to test the role of RAD51 KD incellular survival with olaparib (Selleckchem) treatment. Cellswere plated into 96-well plate with or without doxycycline at 1mg/mL (Sigma-Aldrich). Olaparib was added 72 hours after doxy-cycline induction and then every 3 days for 7 days. A lower dose ofdoxycycline (10 ng/mL) was used to induce 2-fold KD in theSUM149KD cell line.

In vivo tumorigenesis in NOD/SCID miceThe mice were in-house bred and housed in pathogen-free

rodent facilities at the University of Michigan. All supplies (cages,chow, and sterile water) were autoclaved, and all experimentswere conducted according to standard by the University Com-mittee on the Use and Care of Animals. SUM149 and SUM159cells were injected into the mammary fat pads of 6- to 8-week-oldfemale NOD/SCID mice. Olaparib was administrated 15 mg/kgdaily via intraperitoneal injection, and doxycycline (2 mg/mL)was administrated in the water supply [5% sucrose (w/v); Sigma-Aldrich], and tumors were monitored weekly. Animals wereeuthanized by the end of treatment or when the tumors reachedan average of 600 mm3. Tumors were minced and digested by 1Xcollagenase/hyaluronidase (Stem Cell Technologies) in medium199 (Invitrogen), and filtered through a 40-mm nylon mesh. Livetumor cellswere sortedbyfluorescence-activated cell sortingusing

Translational Relevance

Triple-negative breast cancer is an aggressive subtype ofbreast cancer that causes significant morbidity and mortality.Although new chemotherapy options have improved patientoutcomes over the past fewdecades, the development of resist-ance to standard chemotherapy options remains a vexingtherapeutic challenge. Cancer stem cells (CSC) are thoughtto drive this resistance. PARP inhibitors (PARPi) are a prom-ising new therapy in triple-negative breast cancer. Here, wedemonstrate that CSCs are resistant to PARPi and displayelevated RAD51 foci formation efficiency after DNA damage,suggesting that increased efficiency of DNA repair mechanismmight contribute to the PARPi resistance. These findingssuggest that combining PARPiwith inhibition of RAD51 couldlead to improved therapeutic response in patients with triple-negative breast cancer.

RAD51 Mediates Cancer Stem Cell Resistance to PARPi

www.aacrjournals.org Clin Cancer Res; 23(2) January 15, 2017 515




anti-human HLA-A, B, C (BioLegend) and DAPI (Sigma-Aldrich).Tumor cells from each treatment group were implanted at 5,000,500, and 50 cells into each mammary fat pad of NOD/SCIDmouse, and the mice were euthanized when tumors volumereached 600 mm3.

Flow cytometryThe ALDEFLUOR assay was performed according to the man-

ufacturer's (StemCell Technologies) instruction. Dissociated cellswere suspended in assay buffer containing ALEDEFLUOR sub-strate and incubated with or without aldehyde dehydrogenaseinhibitor diethylaminobenzaldehyde. MoFlo Astrios (BeckmanCoulter) and Summit software were used for data acquisition andanalysis.

ImmunostainingFlow-sorted cells grown on chamber slides were fixed in 4%

paraformaldehyde for 10minutes and permeabilized with 0.25%Triton-100. Primary antibodies RAD51 (Santa Cruz Biotechnol-ogy) and gH2AX (Millipore) were applied at room temperaturefor 1 hour followed by Alexa-fluor 488 and 546 (Invitrogen) for30minutes. Cells were thenmounted with ProLong gold antifadereagent with DAPI (Invitrogen).

Statistical analysisOne- and two-way ANOVA test was applied for analyzing in

vitro and in vivo comparisons, and correction for multiple com-parisons was performed using Tukey or Sidak test as appropriate.Image J was used for Western blot quantification. StepOne soft-ware (Applied Biosystems) was used for analyzing real-time PCRdata, and extreme limiting dilution analysis was performed forcalculating CSC frequency (16).

Statement of cell line authenticityHCC1937 and MDAMB231 cells were purchased from the

ATCC, and their identity is routinely monitored by short tandemrepeat (STR) profiling. SUM149 and SUM159 cells were gifts fromDr. Steven Ethier. All cell lines were cryopreserved within 10 pas-sages, and no thawed cell aliquots were cultured for more than6 months continuously. Cells were monitored regularly for myco-plasma contaminationusing theMycoAlertMycoplasmaDetectionKit (Lonza). No cell authentication was performed by the authors.

ResultsCSCs in BRCA1-mutant breast cancer are resistant to PARPinhibition

PARPi have been reported to effectively target BRCA1-mutantbreast cancer cell lines and tumors (2, 3, 17). We therefore testedthe cytotoxic effect of olaparib on four TNBC cell lines includingboth BRCA1-mutant and BRCA1–wild-type cell lines: SUM149,SUM159, HCC1937, andMDA-MB-231, and the status of BRCA1deficiency was confirmed by Western blotting (SupplementaryFig. S1A). As expected, at clinically relevant concentrations, PARPitreatment resulted in fewer viable cells inBRCA1-mutant cell lines(SUM149 and HCC1937) compared with those with wild-typeBRCA1 (SUM159 and MDAMB231; Fig. 1A and B). These dataconfirm the synthetic lethality of PARP inhibition and BRCA1mutation in TNBC.

Accumulating evidence fromanumber of solid tumors suggeststhat CSCs aremore resistant to chemotherapy and radiotherapy as

compared with bulk tumor cells, but there is limited data on theeffect of PARPi on CSC populations. We therefore assessedwhether PARP inhibition targets the CSC population in TNBCcell lines using ALDEFLUOR assay to identify CSCs. Despite thedramatic proapoptotic effect of PARP inhibition on BRCA1-mutant bulk tumor cells, the proportion of CSCs, identified byhigh aldehyde dehydrogenase activity, was elevated after 7 days ofPARPi treatment in BRCA1-mutant cell lines SUM149 andHCC1937 (Fig. 1C).More importantly, the absoluteCSCnumber,which was determined by the total cell number multiplied by thepercentage of ALDEFLUOR-positive cells, was not affected byPARP inhibition (Fig. 1D). PARP inhibition also had limitedeffect on the CSCs in BRCA1–wild-type SUM159 and MDA-MB-231 (Fig. 1C and D), consistent with the expectation thatneither bulk tumor cells nor CSCs are affected by PARPi treatmentbecause of the presence of functional BRCA1. Our data suggestthat although PARPi effectively targets bulk populations inBRCA1-deficient tumor cells, the CSCs in these tumors are resis-tant to PARP inhibition.

Although the mechanisms of PARPi resistance in CSCs are notfully understood, we have previously demonstrated that the CSCpopulation expresses elevated levels of several DNA repair pro-teins (10). We thus performed an exploratory PCR array of 84 keyDNA repair genes. From this array, we identified RAD51, whichassists in DNA double-strand break repair, as having higherexpression in CSCs compared with bulk tumor cells in SUM149cells (Supplementary Fig. S1B). To confirm the protein expressionlevels of RAD51 in CSC and non-CSC populations, SUM149 andSUM159 cells were sorted based on aldehyde dehydrogenaseactivity. Western blot revealed 1.76- and 1.13-fold increases,respectively, in RAD51 protein levels in the CSC populations ofSUM149 and SUM159 compared with the non-stem cells (Fig. 2Aand B). To assess RAD51 localization, sorted cells were seededonto chamber slides and irradiated by 4 Gy single dose of X-rayradiation to induce DNA double-strand breaks, and the extent ofRAD51 foci formation was determined at 0, 3, 12, and 24 hoursafter radiotherapy. ALDEFLUOR-positive SUM149 (BRCA1-mutant) cells displayed a significant increase in RAD51 focistaining at 3, 12, and 24 hours after radiotherapy compared withthe non-CSC population. In contrast, in SUM159 (BRCA1–wild-type), RAD51 foci formation was similar in both CSC and non-CSC compartments (Fig. 2C and D; additional cell line data inSupplementary Fig. S2A). Consistent with previous studies show-ing impaired homologous recombinant DNA repair in cells withBRCA1 mutation (18–20), DNA damage induced much higherproportions of cells with RAD51 foci in the BRCA1–wild-typeSUM159 cell line comparedwith SUM149 cells. In addition, long-term PARPi treatment generated cells with an increase in RAD51expression in TNBCs (Supplementary Fig. S2B). Our data suggesta potential link between RAD51 and olaparib resistance found intheCSCpopulation ofBRCA1-mutant cells, thus targeting RAD51may enhance the efficacy of PARPi.

RAD51 KD sensitizes SUM149 and SUM159 CSCs to PARPinhibitor in vitro

The association between RAD51 and CSCs led us to furtherstudy thismolecule. We infected SUM149 and SUM159 cells witha doxycycline-inducible RAD51 shRNA lentiviral system. Theefficiency of RAD51 KDwas confirmed by real-time PCR of sortedCSCandnon-CSC cells to ensure the efficiencyof silencingRAD51in both populations. Western blot and immunofluorescence foci

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Clin Cancer Res; 23(2) January 15, 2017 Clinical Cancer Research516




stainingwere also performed to ensure KDat protein level (Fig. 3Aand B; Supplementary Fig. S3). RAD51 KD resulted in a 50%decrease (P < 0.05) in the viable cell count in SUM149 cells, likelydue to G0–G1 phase cell-cycle arrest, as we demonstrated a 20%increase of SUM149 cells in G0–G1 phase compared with thosewith RAD51 KD (P < 0.01). However, RAD51 KD showed limitedeffect on SUM159 cell counts. In addition, we demonstrated thatRAD51 KD did not affect CSC populations in both cell lines(Supplementary Fig. S4). To test the hypothesis that RAD51facilitates resistance to PARP inhibition, RAD51 KD SUM149 andSUM159 cells were treated with olaparib. The MTT assay revealedthat RAD51 KD increased sensitivity to the PARPi (P < 0.001) atlow concentrations (10nmol/L and100nmol/L) in SUM149 after7 days of treatment (Fig. 3C). More importantly, a decrease inRAD51 sensitized the CSC population in SUM149 cells to thePARPi treatment, and the effect was dose-dependent (Fig. 3E). Onthe other hand, BRCA1-wild-type SUM159 with functionalRAD51 did not respond to PARP inhibition at 10 nmol/L and100 nmol/L, whereas a 60% decrease in viable cells was inducedby PARPi after RAD51 KD (P < 0.001; Fig. 3D). The SUM159 CSCpopulation also decreased with olaparib treatment in the RAD51KD cells (P < 0.05 and

KD further reduced tumor size by approximately 60% and 75%in early and late treatment groups, respectively. Althoughtumor growth resumed when RAD51 expression was restored,the inhibitory effect of combined treatment was sustained evenafter treatment was terminated (Fig. 4A and B). Tumors fromRAD51 KD and combination treatment groups were 60% and92% smaller (by weight), respectively, when compared withmice with control and were 45% and 87% smaller (by weight),respectively, compared with PARP inhibition alone in earlytreatment (Fig. 4C). In the late treatment groups, RAD51 KDand combined treatment reduced the tumor weight by 60% and80%, respectively, compared with mice receiving vehicle con-trol and PARPi alone (Fig. 4D). Our in vivo results are consistentwith in vitro data suggesting that knocking down RAD51 inhi-

bits SUM149 tumor progression, and the addition of a PARPienhances this effect.

In order to assess the effects of PARP inhibition on the CSCfrequency, serial dilutions of SUM149 xenografts (control andtreated) were re-injected into mouse mammary fat pads. Usingthis gold standard for validating the effect of compounds onCSC population, we were able to calculate the frequency of CSCfrom each treatment group and performed statistical compar-isons using ELDA software as previously reported (16, 21, 22).Compared with control treatment, RAD51 KD and combinedtreatments reduced the CSC frequency by 3 and 10 times,respectively, statistical analysis revealed significant decrease inCSC frequency in combined groups compared to control (P <0.01) and PARPi (P < 0.05) treatments. These results confirm

Figure 2.

RAD51 foci are associatedwith theCSCpopulationofBRCA1-mutant TNBC cells.A,SUM149 and SUM159 cellswere sorted based on aldehydedehydrogenase activity,and protein was gathered for Western blot to test RAD51 expression in different subpopulations. B, Quantification of RAD51 expression level; bar chart isnormalized against b-actin. C and D, sorted ALDEFLUOR-positive and -negative cells were seeded onto glass chamber slides and radiated at 4 Gy single dosethe following morning; cells were then fixed at 0, 3, 8, 12, and 24 hours after radiotherapy and stained with RAD51. Nuclei with � 5 foci were counted asRAD51-positive cells. RAD51 foci scoring in sorted ALDEFLUOR-positive and -negative (C) SUM149 and (D) SUM159 cells. E, Representative RAD51 staining inSUM149 and SUM159 cells after radiotherapy. Bar, 100 mmol/L; a total of 6 fields with >100 cells were counted at each time point from� 3 biological repeats. Mean�SEM; �, P < 0.05 and �� , P < 0.01.

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our hypothesis that RAD51 mediates the sensitivity of CSCs toPARPi.

RAD51 KD sensitizes BRCA1–wild-type SUM159 cells to PARPinhibition in vivo

The sensitization to PARPi mediated by RAD51 KD that weobserved in BRCA1–wild-type SUM159 cells in vitro led us to testwhether we would observe the same effect in an in vivo mousemodel. As described above, SUM159 cells were injected into themammary fat pads of NOD/SCID mice, and treatments werestarted immediately (early treatment) or after the tumors reached

around 15 mm3 (delayed treatment) and continued for 7 weeks.Both RAD51 KD and combination (PARPi and RAD51 KD)treatments slowed tumor progression compared with control(P < 0.05) in early treatment groups (Fig. 5A). This inhibitoryeffect was also seenwhen the treatmentwas given after the tumorswere established (Fig. 5B); however, the decrease was notsignificant.

With regard to CSCs, re-implantation of the SUM159 xenograftcells from the combined treatment in both early and late treat-ment groups failed to form any tumor. This indicates that thestrategy of combined RAD51 inhibition and PARPi is able to

Figure 3.

RAD51 KD sensitizes TNBC cells to PARPi. A, RAD51 shRNA was induced in SUM149 and SUM159 cells for 3 days, and the cells were harvested for ALDEFLUORsorting, and sorted subpopulations were lysed for qRT-PCR. B, RAD51 KD was induced for 3 days, and protein was collected for Western blot. C, SUM149and (D) SUM159 cells with/without RAD51 KD were plated into 96-well plate at the same density, and MTT was performed after 7 days with olaparib treatment at0, 10, and 100 nmol/L. E and F, Aldehyde dehydrogenase activity was also assessed after 7 days of olaparib treatment (0, 10, and 100 nmol/L) in (E) SUM149and (F) SUM159 cells with/without RAD51 KD. Mean � SEM from � 3 biological repeats; � , P < 0.05; �� , P < 0.01; and �� , P < 0.001.






eradicate the tumor-initiating cell population. The CSC frequencyof combined treatment decreased significantly comparedwith thecontrol (P < 0.05), PARP inhibition (P < 0.001 and P < 0.01 inearly and late treatment groups, respectively), andRAD51KD (P <0.01) groups (Fig. 5C and D). Together, these results indicate thatour therapeutic regimen targets the BRCA1–wild-type SUM159cells (both CSC and bulk-tumor populations), supporting our invitro findings of the involvement of RAD51 in sensitivity ofBRCA1–wild-type SUM159 cells to the PARPi olaparib, andimportantly, it suggests an effective therapeutic intervention fortargeting TNBC.

DiscussionDespite the promising results of PARPi in treating TNBCs with

BRCA deficiency, resistance is still a significant issue. The over-whelming evidence of the importance of CSCs to drug resistanceledus to study the effect of PARP inhibition onCSCs. In this study,we used in vitromodels andmouse xenografts to demonstrate theimportance of RAD51 in PARPi resistance of CSCs in BRCA1-deficient and–wild-type TNBCs.Our studywas limited to four celllines due to the availability of BRCA-mutated breast cancer cellslines (23); however, we have also provided evidence that RAD51plays a role inmediating resistance to PARPi in basal and claudin-low TNBC.

Sensitivity to PARP inhibition in cells that are deficient incomponents of the homologous repair pathway suggests a

broader clinical potential for PARPi in treating triple-negativebreast tumors (2). However, our results showing a relativelymodest influence of PARPi on CSCs of both BRCA1-mutant andBRCA1–wild-type TNBC cells identify a potential mechanismfor PARPi resistance. Although overexpression of PARP1 hasbeen found to be associated with PARPi-resistant ALDEFLUOR-positive breast cancer cells (24), the molecular mechanismsmediating CSC resistance to PARP inhibition in TNBCs are notknown. Here, we have shown that RAD51 is involved in thisresistance.

The synthetic lethality of PARP inhibition in TNBCs is based onBRCA1 deficiency; however, the CSCs with higher RAD51 expres-sion exhibited reduced sensitivity toward PARP inhibition. Inaddition, even among the TNBC cells with BRCA1 mutation,HCC1937 displayed less sensitivity toward the PARPi comparedwith SUM149, likely due to higher basal RAD51 level. Our resultsare consistentwith previousfindings showing that overexpressionof RAD51 compensates for BRCA1 deficiency (25), thereforeallowing the homologous recombinase pathway to repair DNAdamage when PARP is inhibited. In addition, PARPi-resistantBRCA1-mutant TNBC cells, selected by long-term treatment withPARPi, exhibited elevated RAD51 activity comparedwith parentalcells (26), supporting our observation of RAD51 in mediatingsensitivity to PARPi. We have also examined the expression ofRAD51 upstream regulators: BRCA1 and BRCA2 in CSC and non-CSC subpopulations and detected no significant differences inexpression in these cell populations (data not shown). However,

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RAD51 KD 11716/62/60/6 RAD51 KDControl 0.105Combined 35996/60/60/6 RAD51 KDPARPi 0.624

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Figure 4.

CSCs of BRCA1-mutant SUM149become sensitive to PARPi whenRAD51 is absent. A–D, 50,000 SUM149cells were implanted into themammary fat pad of 6- to 8-week-oldfemale NOD/SCID mice and 4 groupsof treatment: vehicle control,PARPi alone, RAD51 KD alone,PARPiþRAD51 KD were given either(A) immediately after implantation or(B) when tumors reached 2 to 3 mm indiameter for a total of 8 weeks, andtumor size was monitored weekly.Mice were sacrificed by the end oftreatment, and treatments werewithdrawn from those with smalltumors until they reached 10 to 15 mmin diameter. C and D, Tumor weightwas measured and recorded by theend of experiment for early and latetreatment groups. Mean � SEM,n ¼ 5. E, Tumors cells from latetreatment groups were collected andre-injected to the mammary fat pad ofNOD/SCID at 50, 500, and 5,000density per treatment for limitingdilution assay. CSC frequency andstatistical analysis were calculatedusing ELDA software. �, P < 0.05;�� , P < 0.01; ��, P < 0.001; and�� , P < 0.0001.

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we cannot rule out involvement of other molecules upstream ofRAD51 in mediating PARPi resistance.

Overexpression of RAD51 has been linked with therapy resis-tance, and CSCs are known to be more resistant to chemotherapyand radiotherapy compared with non-CSCs. Inmouse embryonicstem cells, RAD51 protein is expressed at very high levels com-pared with differentiated cells (27); and human embryonic stemcells exhibit more efficient DNA damage repair compared withprimary fibroblasts (28). In cancers, increased levels of RAD51were found in invasive pancreatic cancer cells displaying CSCproperties, and higher expression was also associated with aggres-siveness of primary tumors (13). The CSCs of MDA-MB-231repaired DNA damage more efficiently than the non-CSC cellsafter irradiation, as evidenced by a much higher expression ofRAD51 foci (29). The factors regulating RAD51 expression inCSCs remain unknown. In addition to BRCA1/2, RAD51 is alsolinked to ERK1/2, miR-96, and hypoxia (30–32). The MEK/ERKsignaling pathway has been found to facilitate tumor growth andangiogenesis of TNBC in mouse models (33); miR-96 enhancedbreast cancer cell proliferation and anchorage-independentgrowth (34), while hypoxia was shown to promote CD44þ

/CD24� expansion (35). Interestingly, miR-96 and hypoxia actu-ally downregulate RAD51 expression, suggesting that CSCs ofTNBC cells are RAD51 independent, which is consistent with ourdata that knocking down RAD51 alone did not reduce CSCfrequency. However, RAD51 KD did slow down tumor progres-sion, and this inhibitory effect is reversible when the expressionwas rescued, suggesting that RAD51 is involved in regulating CSCproliferation.

PARP inhibitions have shown promising results in subsets ofTNBC patients that manifest deficiency in DNA damage repair.Our findings of the role of RAD51 in PARPi resistance in CSCs of

BRCA1-mutated TNBCs and BRCA1–wild-type TNBCs suggestthat resistance to PARPi may be overcome by targeting both CSCsand bulk-tumor cells. Furthermore, by targeting RAD51, itmay bepossible to greatly expand the sensitivity of TNBCs to PARPi,beyond thosewith defective BRCA1proteins. This novel approachholds potential for significantly improved therapies for TNBC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Liu, M.L. Burness, S. Bai, T.K. Luther, M.S. Wicha,S. LiuDevelopment of methodology: Y. Liu, M.L. Burness, R. Martin-Trevino, S. Bai,T.K. Luther, M.S. Wicha, S. LiuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Liu, M.L. Burness, R. Martin-Trevino, J. Guy,R. Harouaka, M.D. Brooks, A. Fox, T.K. Luther, A. Davis, T.L. Baker, J. Colacino,M.S. Wicha, S. LiuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Liu, M.L. Burness, J. Guy, R. Harouaka, A. Fox,J. Colacino, S.G. Clouthier, S. LiuWriting, review, and/or revision of the manuscript: Y. Liu, M.L. Burness,T.K. Luther, S.G. Clouthier, M.S. Wicha, S. LiuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Liu, M.L. Burness, R. Martin-Trevino,R. Harouaka, L. Shang, T.K. Luther, Z.-m. Shao, S. LiuStudy supervision: M.L. Burness, S.G. Clouthier, M.S. Wicha, S. Liu

AcknowledgmentsThe authors thank the University of Michigan Flow Core and Vector Core for

excellent technical assistance. They also thankDrs. Felix Feng,Ming Luo, and JillGranger for their comments during article preparation, and Drs. Guan-TianLang, Xin Hu, Dafydd Thomas, and Sofia Merajver for assistance in acquiringclinical specimens.

C

D

Early treatment

groups

Limiting dilutionsTumors/implantation

CSCfrequency(1 in/…)

PGroup 2Group 1ControlCombined 0.0172*PARPiCombined500050050 0.000261***

Control 34042/31/30/3 RAD51 KDCombined 0.00137**PARPi 7863/32/40/3 PARPiControl 0.106

RAD51 KD 11713/31/30/3 RAD51 KDControl 0.257Combined Inf.0/30/30/3 RAD51 KDPARPi 0.697

Latetreatment

groups

Limiting dilutionsTumors/implantation

CSCfrequency(1 in/…)

PGroup 2Group 1ControlCombined 0.0172*PARPiCombined500050050 0.00513**

Control 34042/31/30/3 RAD51 KDCombined 0.0101**PARPi 23742/32/30/3 PARPiControl 0.658

RAD51 KD 31891/33/30/3 RAD51 KDControl 0.934Combined Inf.0/30/30/3 RAD51 KDPARPi 0.693

A B

Figure 5.

RAD51 KD sensitizes BRCA1–wild-typeSUM159 cells to PARPi. A and B, 50,000SUM159 cells were injected into themammary fat pad of NOD/SCID mice andtreatments: vehicle control, PARPi alone,RAD51 KD, PARPiþRAD51 KD were given(A) immediately after implantation or (B)when tumors reached 2 to 3mm in diameterfor 7 to 8 weeks. Mean � SEM, n ¼ 5. C andD, Tumors were harvested by the end oftreatment and processed into single cellsand implanted to second recipient at 50,500, and 5,000 cells per mammary fat padfor serial dilution assay. CSC frequency andstatistical analysis were calculated usingELDA software. � , P < 0.05; �� , P < 0.01; and�� , P < 0.001.






Grant SupportThis work was supported by the DOD grant (W81XWH-12-1-0147), CAS

stem cell grant XDA01040410, NSFC grants 81472741 and 81322033 (toS. Liu), The Dr. Frank Limpert Clinical Scholar Award and NSFC grant81530075 for the tumor block cutting (to M.L. Burness), Susan G. Komen forthe Cure Promise Award (KG120001), Breast Cancer Research FoundationAward (N015445), Senior Taubman Scholar Award, and Cis Maisel gift tosupport CSC research (to M.S. Wicha).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 17, 2015; revised July 29, 2016; accepted August 1, 2016;published OnlineFirst December 29, 2016.

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Liu et al.




2017;23:514-522. Published OnlineFirst December 29, 2016.Clin Cancer Res Yajing Liu, Monika L. Burness, Rachel Martin-Trevino, et al. Inhibition in Triple-Negative Breast CancerRAD51 Mediates Resistance of Cancer Stem Cells to PARP

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